The goal of this work is the elucidation of the mechanisms controlling the rate of protein synthesis at the cytoplasmic level in mammalian cells and the differences between these mechanisms in normal and malignant cells. We have demonstrated that, upon nutrient deprivation (essential amino acid, glucose, or serum) of the Ehrlich ascites tumor cell in suspension culture, one such mechanism modulates chain initiation by inhibiting the formation of the complex between the 40s ribosomal subunit and the Met-tRNAf.eIF-2.GTP ternary complex (i.e., the 40s initiation complex). We are now examining eIF-2 function. During the initiation reactions, GTP in the ternary complex is hydrolyzed and eIF-2 is released as, or rapidly forms, eIF-2.GDP. We have found a GDP/GTP exchange factor (GEF) that allows GTP to replace GDP in the binary complex. This is a new initiation factor, since without GEF the half-time of dissociation of the eIF-2.GDP complex is greater than 30 min. Phosphorylation of the alpha subunit of eIF-2 prevents the GEF-catalyzed GDP exchange and thus can stop eIF-2 cycling and chain initiation. We have measured the alterations in the extent of phosphorylation of eIF-2 in cells deprived of an essential amino acid. We find less than 10% change in extent of phosphorylation of total eIF-2 and of the subfraction of eIF-2 bound to ribosomes, bound to free 40s ribosomal subunits, and soluble eIF-2. We are presently developing approaches to determine whether these changes can account for the large changes seen in the rate of protein synthesis. Over the past year, we have found that small changes in ADP:ATP and GDP:GTP ratios, and not the absolute amount of any of these nucleotides, result in large changes in the rate of polypeptide chain initiation in Ehrlich cell-free protein-synthesizing systems. Currently, we are investigating the effect of altering these nucleotide ratios in the cell-free system on eIF-2 function and phosphorylation. We have also found that defective initiation--in cell-free protein-synthesizing systems prepared from heat-shocked Ehrlich cells--is completely restored by the addition of eIF-4F (mRNA cap binding protein). (F)